And Matrona Basilaris Selys, 1853 (Odonata: Calopterygidae)

Zootaxa 4306 (4): 580–592 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2017 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4306.4.8 http://zoobank.org/urn:lsid:zoobank.org:pub:3EC84E84-06D7-4B0C-8766-92842F768FED

Descriptions of larvae of Vestalaria venusta (Hämäläinen, 2004) and Matrona basilaris Selys, 1853 (Odonata: Calopterygidae)

RUNXI WANG1, XIN YU1, 2, JUNLI XUE1 & XIN NING1 1Insititute of Entomology, College of Life Sciences, Nankai University, Tianjin, 300071, China 2Corresponding author. E-mail: [email protected]

Abstract

Larva of Vestalaria venusta is identified using DNA barcoding match with the adult and described in the first time. Mor- phological characters are compared with those of Matrona basilaris and Vestalis amoena. The validity of genus the Ves- talaria is reconfirmed. The important role of DNA barcoding in odonate larva identification is emphasized.

Key words: Zygoptera, Vestalaria, Matrona, Vestalis, larva, DNA barcode

Introduction

Vestalaria May, 1935, a small genus of Calopterygidae, is restricted to south China and Vietnam and includes five known species: V. m iao (Wilson & Reels, 2001), V. smaragdina (Selys, 1879), V. velata (Ris, 1912), V. venusta (Hämäläinen, 2004), V. vinnula Hämäläinen, 2006 (Hämäläinen 2016, Schorr & Paulson 2017). The genus Vestalaria was recently reinstated as separate from Vestalis Selys, 1853 by Hämäläinen (2004, 2006) according to careful morphological studies on adults and this received further support from a molecular study (Guan et al. 2012). No larva of Vestalaria has been described yet. DNA barcoding as a practical tool for associating larvae and adults of insects (Hebert et al. 2004), has been increasingly used in Odonata (Orr & Dow 2015, Steinhoff et al. 2016, Yu 2016). It has been confirmed that the combined nuclear gene of the ribosomal ITS1–5.8S–ITS2 region (ITS) is quite stable and reliable in molecular taxonomy at the genus and species level in Odonata, especially in Calopterygidae (Guan et al. 2012, Yu et al. 2015). In this study we use DNA barcoding, based on ITS sequence, to identify and describe a larva of genus Vestalaria. It is compared morphologically with Matrona and Vestalis larvae.

Material and methods

Samples. Calopterygidae adults and larvae were sampled from three localities (cf. Taxonomic account). Adults were caught with a standard insect net. Larvae were collected using a dip net and manual removal of stones. Attempts were made to rear the larvae in plastic containers but they died en route to the laboratory. Photographs of living specimens were taken in the field, before or just after collection, with a digital camera (Nikon D3200, Thailand). Photographs of detailed morphological features were taken in the laboratory using a Zeiss V20 microphotography system. Specimens were preserved in absolute ethanol and were examined and dissected under a Zeiss V8 stereomicroscope. Larvae from all localities were morphologically compared to each other and to description of the larva of Vestalis amoena Selys, 1853 given in Lieftinck (1965). As V. amoena larvae were not available for dissection and direct comparison to the sampled material in this study, this species was left out of the morphological characterization given below. Watson (1956) terminology of larva mandible description was used. At the time of collecting in 2011 (cf. Taxonomic account) the second author (XY) found a little stream where

580 Accepted by M. Marinov: 29 Jun. 2017; published: 18 Aug. 2017 the rare species Matrona oreades Hämäläinen, Yu & Zhang, 2011 was very abundant – both tenerals and sexually mature individuals. As no other calopterygid species was found around the stream (except a few of Archineura incarnata (Karsch, 1891) and one teneral male of V. venusta from more than 50 meters away) the larvae were provisionally identified as the dominant adult at the site M. oreades. However, according to subsequent molecular analysis published in Yu et al. (2015), the larvae were not Matrona but closely related to Vestalis, thus necessitating a special study of the larvae. In order to confirm the identification and classification of the larvae, ITS sequences from a total of 23 specimens were selected for the phylogenetic analysis (Table 1). Those sequences included material of the V. venusta and Matrona basilaris Selys, 1853 larvae described morphologically below in addition to already deposited nine sequences of Vestalis and Vestalaria species in NCBI (National Centre for Biotechnology Information, https://www.ncbi.nlm.nih.gov/) and 11 sequences of Matrona species used in Yu et al. (2015). Note, that Yu et al. (2015) utilised the four M. basilaris used for the morphological description of larvae in this study. Voucher materials of all specimens except those from NCBI were deposited in the Institute of Entomology, College of Life Sciences, Nankai University, China. DNA extraction and sequencing. Total genomic DNA was isolated from hind leg muscle samples using UniversalGen DNA Kit (Beijing ComWin Biotech). Small doses were used as a template for polymerase chain reaction (PCR) amplification. PCR was carried out in 40 µL final reaction volumes containing 20µL of 2×Es Taq MasterMix (Beijing ComWin Biotech), 15.5µL ddH2O, 1.5µL of each primer and genomic DNA. For larvae samples, the DNA fragments of nuclear internal transcribed spacer (ITS) were amplified with the primers Vrain2F (5’-CTT TGT ACA CAC CGC CCG TCG CT-3’) and Vrain2R (5’-TTT CAC TCG CCG TTA CTA AGG GAA TC-3’) (Dumont et al. 2010). For the adult Vestalaria venusta, ITS 1 and ITS 2 were amplified with the following primers respectively: 5’-GGC CAA ACT TGA TCA TTT AG-3’ and 5’-GCC GGC CCT CAG CCA G-3’ for ITS1, 5’-CGG TGG ATC ACT CGG CTC GT-3’ and 5’-TTT CAC TCG CCG TTA CTA AGG GAA TC-3’ for ITS2 (Futahashi & Sasamoto 2012). The PCR cycling procedure was 2 min at 95°C followed by 35 cycles of denaturation at 95°C for 30s, annealing temperature at 56°C for 30s, and extension at 72°C for 1 min, with a final single extra extension step at 72°C for 8min (Larvae specimens). Annealing temperature 46.5°C for ITS1, 56.5°C for ITS2 for the adult specimen. All PCR products were visualized via 1% agarose gel electrophoresis and amplifications were purified using a gel extraction kit (Sangon Biotech, Shanghai), then sent to commercial companies (BGI, Beijing) for sequencing based on Sanger’s chain termination method. All fragments were sequenced in both directions. DNA analysis. All sequences were edited, assembled and aligned in BioEdit v7.2.0 (Hall 1999). Alignments of protein coding genes were translated to amino acids using MEGA v6.06 (Tamura et al. 2013) to detect frameshift mutations and premature stop codons, which may indicate the presence of pseudogenes. Sequences were aligned using the ClustalX version 2.1 program package (http://www.clustal.org/) with default settings, and subsequently corrected manually in terms of the sequence chromatogram to ensure each mutation loci was credible. Subsequent analyses were performed using optimality criteria including maximumlikelihood (ML) and the Bayesian inference algorithm (BI) to resolve the phylogenetic relationships. ML analyses were derived using MEGA v6.06 General Time Reversible model, predicted through ModelTest3.7 (Posada & Crandall 1998). BI analysis was performed using MrBayes v3.1.2 (Huelsenbeck & Ronquist 2001) under GTR + I + G model implied by MrModeltest v2.3 (Nylander 2004). All the acquired trees were set to 1 million generations and for every 1000 generations the chain was sampled. Generations with standard deviation values higher than 0.01 were burned. Trees were displayed and rendered with FigTree v1.4.0 (Rambaut 2012).

Results

Based on the 23 sequences of ITS, all the phylogenetic trees (ML, BI) produced the same topological results, which strongly supported (BPP=0.84, MLB=95) the larvae and the adult V. venusta to form a monophylum (Fig. 1). Vestalaria was also recovered as a monophylum (BPP=1.00, MLB=100) with Vestalis as sister group with very strong support (BPP=1.00, MLB=100). In the Matrona clade, M. basilaris together with M. mazu Yu, Xue & Hämäläinen, 2015 formed a monophylum (BPP=1.00, MLB=95) with M. oreades as sister (BPP=1.00, MLB=100). This result indicated that the larvae involved here are the same species with the adult, viz. V. venusta. The validity of genus level of Vestalaria was confirmed again by molecular phylogeny.

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582 · Zootaxa 4306 (4) © 2017 Magnolia Press WANG ET AL. FIGURE 1. Phylogenetic reconstruction based on ITS. Values of Bayesian posterior probabilities and Maximum likelihood bootstrap are indicated at nodes respectively, the adult and larvae of Vestalaria venusta are in red colour.

Taxonomic account

Vestalaria venusta Hämäläinen, 2004

Material studied. Adult 1 ♂ (SCYA08) and larvae 1 ♂ (SCYA05), 1 ♀ (SCYA06): China, Sichuan, Yaan, Bifengxia Town, Houyan Village (103.0472° E, 30.1019° N), 25-vii-2011, Xin Yu leg. Description of larva. Diagnosis. A slender zygopteran with a relatively small head, long legs with distinct bands, and long abdomen with long sword-like gills. Ground colour of body pale brown (Fig. 2a). Head. Relatively small; in dorsal view general shape a compressed pentagon. Postocular lobes somewhat swollen, terminating in a pair of small dorsal upward-directed protuberances. Antennae 7-segmented, long and robust, especially segment 1 which is very strong and almost twice as long as all other segments together. Segment 2–7 gradually short and tapered from base to apex (Figs. 3a, c). Prementum elongate, strongly expanded distally, about 1.5 times as long as broad, anterior margin deeply cleft with a pair of knife-like projections obliquely truncated apically, each bearing three pairs of setae, the most basal pair only one third as long as the middle pair which is not obvious (Figs. 4a–b). Labial palp robust with three strong, long and incurved distal teeth, of which the middle one is the longest; inner margin of the palp slightly produced, bearing two pairs of setae; movable hook very long and robust (Figs. 4a–b). Maxilla 1.5 times as long as broad; galea and lacinia partly fused; lacinia terminating in four long sharp spines, forming a curved, inward-directed, pitchfork-like structure; galea with three shorter spines directed upward. Palp with short basal segment and a single long banana-shaped terminal segment, reaching to half of most distal spines on galeo-lacinia, covered in dense long hairs (Figs. 5a–d). Right mandible (Figs. 6a–b) with four long and well developed incisors and a fifth innermost tooth; third incisor is the longest; molar crest produced to form two well-defined curved bifid spine (R 1’1234 y ab, 1’ < 1 < 2 < 4 < 3, a < b). Left mandible (Figs. 6c–d) with four shorter incisors; molar crest produced straight, distal edge serrated with even dense tiny teeth between a and b (L 1’1234 0 a (m 1-11) b, 1’ < 1 < 2 < 4 < 3, a < b) of which it was difficult to determine the exact number. Thorax. Prothorax not strong, narrower than both head and meso- and metathorax; meso- and metathorax almost rectangular in shape. Legs long and progressively slightly longer from pro- to metathorax. Femur slightly

LARVA OF VESTALARIA VENUSTA Zootaxa 4306 (4) © 2017 Magnolia Press · 583 longer than tibia, each with two well defined dark bands. Wing pads narrow and not divergent, moderate long, just exceeding proximal edge of S4 (Fig. 2a). Abdomen. Slender, elongate. Gonapophyses small in male, projecting from middle of S9 to the two-thirds of this segment (Fig. 7a). In female outer gonapophyses projecting from middle of S9 to end of S10; inner gonapophyses reaching still a little further (Fig.7b). Cerci wholly concealed by caudal gills in ventral view (Figs. 7a–b). Paired, long triquetral lateral gills and single lamellate middle gill both bearing tiny setae-like spines along margins (Figs. 8a–b). Lateral gills with sharp apex always longer than the middle which has a nearly rounded apex, and both marked with dark spots (Figs. 9a–b). Microhabitat and behaviour. Larvae were found in a small montane stream with shallow water and stony substrates (Fig. 10). Meanwhile, adults of M. oreades, Anisopleura qingyuanensis Zhou, 1982, Archineura incarnata and Sympetrum eroticum (Selys, 1883) were present around the site in moderate numbers. The larvae were usually found concealed under stones or gravel in the day time and active at night.

FIGURE 2. Larval habitus of: (a) Vestalaria venusta and (b) Matrona basilaris.

Matrona basilaris Selys, 1853

Specimens studied. Larvae: 1 ♂ (AHLA05), 1 ♀ (AHLA14), China, Anhui, Liuan, Jinzhai (115.9585° E, 31.5519° N), 1-v-2015, Hongqu Tang leg.; 2 ♀# (AHLA09, AHLA13), China, Anhui, Liuan, Jinzhai, Tiantangzhai (115.7875° E, 31.1362° N), 2-v-2015, Xin Yu leg. Description. Diagnosis. A slender zygopteran with a depressed and small head, long legs with distinct bands, long abdomen with long sword-like gills. Ground colour of body dark brown (Fig. 2b) but much varied between individuals.

584 · Zootaxa 4306 (4) © 2017 Magnolia Press WANG ET AL. FIGURE 3. Lateral and dorsal view of top of the head: (a, c) Vestalaria venusta; (b, d) Matrona basilaris. Protuberances on postocular lobes are been indicated with red arrows.

Head. Flattened above, widest across the eyes; in dorsal view general shape a compressed pentagon. Postocular lobes little developed so hind margin of head very narrow at base; indistinct (in dorsal view) prouberances about halfway between eye and posterior angle. Antennae 7-segmented, long and robust, with strong and long segment 1 which is almost as twice as remaining segments combined, and short segment 2–7 tapering from proximal to distal (Figs. 3b, d). Prementum elongate, strongly dilated distally, about 2 times as long as broad; anterior margin strongly cleft and forming a pair of knife-like prominences, each prominence bearing three pairs of setae, the most basal pair as long as the middle pair (Fig. 4c). Labial palp robust with three strong, long, and incurved distal teeth, of which the middle one is the longest; movable hook very long and robust with two setae on its base (Fig. 4c). Maxilla 1.5 times as long as broad; galea and lacinia partly fused; lacinia terminating in four long sharp spines, forming a curved, inward-directed, pitchfork-like structure; galea with three shorter spines directed upward. Palp with short basal segment and a single long banana-shaped terminal segment, almost reaching to the end of the most distal spines on galeo-lacinia, covered in dense long hairs (Figs. 5e–h). Right mandible (Figs. 6e–f)

LARVA OF VESTALARIA VENUSTA Zootaxa 4306 (4) © 2017 Magnolia Press · 585 with four rather long and well developed incisors and a fifth innermost tooth; molar crest is greatly reduced (R 1’1234 y ab, 1’ < 1 < 2 < 4 < 3, a < b). Left mandible (Figs. 6g–h) with four shorter incisors; molar crest produced straight, distal edge serrated with 11 fine cusps (L 1’1234 0 a (m1-9) b, 1’ < 1 < 2 < 4 < 3, a < b). Thorax. Prothorax weak, narrow than both meso- and metathorax; meso- and metathorax almost rectangular in shape. Legs long and progressively slightly longer from pro- to metathorax. Femur slightly longer than tibia, each with two distinct dark bands. Wing pads not divergent, narrow, moderate long, just exceed middle of S3 (Fig. 2b).

FIGURE 4. Dorsal view of prementum and labial palps: (a) Vestalaria venusta, right prominence broken; (b) V. venusta, outer distal teeth of left labial palp broken; (c) Matrona basilaris. Positions of lost setae were indicated with little arrows, black ones for long setae and red for short.

FIGURE 5. Maxilla of Vestalaria venusta: (a) left, posterior surface; (b) right, posterior surface; (c) left, innter surface; (d) right, innter surface. Maxilla of Matrona basilaris: (e) left, posterior surface; (f) right, posterior surface; (g) left, innter surface; (h) right, innter surface.

586 · Zootaxa 4306 (4) © 2017 Magnolia Press WANG ET AL. FIGURE 6. Mandible of Vestalaria venusta: (a) right, posterior surface; (b) same, innter surface; (c) left, posterior surface; (d) same, innter surface. Mandible of Matrona basilaris: (a) right, posterior surface; (b) same, innter surface; (c) left, posterior surface; (d) same, innter surface.

Abdomen. Slender, elongate. Gonapophyses very small in male, projecting from middle of S9 to the two-thirds of this segment (Fig. 7c). In female outer gonapophyses projecting from anterior margin of S9 almost to the end of S10; inner gonapophyses as long as the outer (Fig. 7d). Cerci wholly concealed by caudal gill in ventral view (Figs. 7c–d). Paired, long triquetral lateral gills and single lamellate median gill both bearing with setae-like spines along margins and blunt apexes (Figs. 8c–d). Lateral gills longer than the middle and both marked with dark spots (Figs. 9c–d).

FIGURE 7. Gonapophyses in ventral view: (a) Vestalaria venusta male with caudal gills removed to show cerci; (b) V. v enusa female; (c) Matrona basilaris male with lateral caudal gills removed to show cerci; (d) same female with caudal gills removed to show cerci.

Microhabitat and behaviour. Larvae were found both in small montane streams with sandy substrates and open rivers with stony substrates (Figs. 11–12). Some even occurred in very small puddles covered with dense vegetation formed by discontinuous streams. They were usually concealed among stones and gravel, or in water plants, depending on their body colour. The habitat usually had many adults of Mnais tenuis Oguma, 1913.

LARVA OF VESTALARIA VENUSTA Zootaxa 4306 (4) © 2017 Magnolia Press · 587 FIGURE 8. Distal part of caudal gills in lateral view: (a) lateral gill of Vestalaria venusta; (b) middle gill the same; (c) lateral gill of Matrona basilaris; (d) middle gill the same.

FIGURE 9. Caudal gills in lateral view: (a) Vestalaria venusta middle; (b) same lateral; (c) Matrona basilaris middle; (d) same lateral.

FIGURE 11. Habitat of the larva of Matrona basilaris in Liuan, Anhui. Small montane streams with sandy substrates.

LARVA OF VESTALARIA VENUSTA Zootaxa 4306 (4) © 2017 Magnolia Press · 589 Diagnosis. Generally, the larvae of Vestalaria and Matrona have similar appearance, such as a slender body, small head, long and banded legs, and elongate sword-like caudal gills. However, they can be separated easily by several characters listed below (Table 2).

TABLE 2. Comparison between larvae of Vestalaria venusta and Matrona basilaris. V. venusta M. basilaris Head in dorsal view broader posteriorly (Fig. 3c) narrower posteriorly (Fig. 3d) protuberances on postocular lobes of head at the posterior corner (Fig. 3c) at the middle of each side (Fig. 3d) length of the middle cleft of prementum less than palp (Figs. 4a–b) twice length of palp (Fig. 4c) setae on apical processes of prementum three pairs (Figs. 4a–b) two pairs (Fig. 4c) median caudal gill lamellate (Fig. 8b) triquetral (Fig. 8d) apexes of lateral gills sharp (Fig. 8a) blunt (Fig. 8c)

FIGURE 12. Habitat of the larva of Matrona basilaris in Liuan, Anhui. Open rivers with stony substrates.

Discussion

According to Lieftinck (1965), compared with Matrona, larvae of Vestalaria venusta are more like those of Vestalis amoena (Fig. 13a). However, they can be distinguished by the characters of the caudal gills and the number and arrangement of setae on the prementum. In Vestalis the caudal gills are all lamellate type with the lateral gills bearing a prominent mid-rib outside (Fig. 13c), whereas in Vestalaria only the median gill is flat, lateral gills are triquetral in cross-section with rows of short tubercular spines at the ridges (Fig. 8a). Furthermore, the lateral gills

590 · Zootaxa 4306 (4) © 2017 Magnolia Press WANG ET AL. of Vestalaria are definitely longer than the middle with the apexes more acuminate than those of Vestalis (Fig. 13c). According to Lieftinck (1965) there are two pairs of setae on the processes of the prementum in V. amoena, both on the inner margins (Fig. 13b). Vestalaria venusta has three pairs of setae with the upper pair at the middle of the processes and the other two pairs on the inner margins. The bottom pair setae are tiny, not easily seen (Figs. 4a–b). It is worth noting that the setae on the processes of the prementum drop off very easily. However the actual number and position of the setae can still be confirmed from their sockets. The present work again demonstrates the great value of DNA barcoding in larvae identification, especially to those sympatric with congeners and difficult to breed.

FIGURE 13. Figures of Vestalis amoena modified from Lieftinck (1965): (a) habitus; (b) dorsal view of prementum and labial palps; (c) middle (upper) and left lateral (lower) caudal gill.

We thank Dr. M. Hämäläinen for helping to confirm the species of the adult specimen and Dr. Hongqu Tang for providing specimens. We are grateful to Dr. A.G. Orr and Dr. M. Marinov reviewed the manuscript. This project was supported by the National Natural Science Foundation of China (No. 31572299) and the grant of Ministy of Science and Technology of China (No. 2015FY210300).

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